Despite its essential role in supporting life, oxygen can be highly damaging to an organism under certain conditions. For plants, the inadvertent production of active oxygen species (e.g., O2-, H2O2, hydroxyl radicals, and singlet oxygen) occurs as a consequence of normal photosynthetic activity. Exposure to many abiotic stresses can exacerbate the production of active oxygen species, including cold, drought, salt, or high light. The production of active oxygen species near the photosynthetic machinery can result in substantial damage and thus reduce photosynthetic capacity or, under severe conditions, lead to death of the organ or entire plant. Active oxygen species can be produced in other cellular compartments including the mitochondria which themselves have substantial electron transport activity as well as in peroxisomes during the oxidation of glycollate. Active oxygen species are produced in response to attack by many pathogens. Nevertheless, not all active oxygen species are produced by plant processes or responses. Active oxygen species can invade a plant when it is exposed to pollutants such as ozone. However, the production of active oxygen species is not always inadvertent. Active oxygen species can play an important role as signaling molecules. For example, oxygen photoreduction (the Mehler peroxidase reaction) results from the transfer of electrons from photosystem I (PSI) to oxygen to form superoxides which disproportionates to hydrogen peroxide (H2O2), a reaction that is catalyzed by superoxide dismutase. The Mehler reaction thus serves to maintain electron flow through PSI and maintains its correct function. H2O2 acts as a signaling molecule involved in many stress and defense responses (Van Breusegem et al., 2001) and in guard cells can induce stomatal closure (Pei et al., 2000; Zhang et al., 2001). Consequently, plants have had to evolve mechanisms to limit the deleterious effects of many active oxygen species and simultaneously use the production or exposure of certain active oxygen species as information about alterations to the internal or external environment of the plant to mount correct responses to its current conditions. Plants, like most organisms, rely on an array of antioxidants to detoxify active oxygen species.
Of the antioxidants found in plants, ascorbic acid (“ASC”) is the most abundant and is present in millimolar concentrations that range from 10 to 300 mM (Smirnoff, 2000). Glutathione, for example, the other major soluble antioxidant, is typically present at only 10% of the concentration of ASC (Noctor and Foyer, 1998). In its antioxidant role, ASC is used by ascorbate peroxidase to convert H2O2 to water and ASC can directly scavenge superoxide, hydroxyl radicals, and singlet oxygen. ASC also contributes to the regulation of the cellular redox state and is used to regenerate a-tocopherol from a-tocopherol radicals that are produced from the reduction of lipid peroxyl radicals. ASC can serve as an enzyme co-factor, e.g., for violaxanthin de-epoxidase (VDE) (Eskling et al., 1997) which catalyzes the conversion of violaxanthin to zeaxanthin (the Xanthophyll cycle), which is required for the dissipation of excess excitation energy during non-photochemical quenching. ASC is also involved in the regulation of cell elongation and progression through the cell cycle (reviewed in Horemans et al., 2000). This partial list of cellular functions demonstrates the importance of ASC to the health and growth of the cell, and ultimately, the plant.
ASC biosynthesis differs from that in mammals and has been shown to result from the oxidation of L-galactose to L-galactono-1,4-lactone which in turn is oxidized to ASC by L-galactono-1,4-lactone dehydrogenase. Although most of the biosynthetic pathway is carried out in the cytosol, the final step occurs at the inner mitochondrial membrane where the L-galactono-1,4-lactone dehydrogenase is located (Siendones et al., 1999; Bartoli et al., 2000). Feedback inhibition of ASC synthesis by the ASC pool size has been demonstrated (Pallanca and Smirnoff, 2000). Given that ASC is present in most compartments of the cell including the mitochondria, cytosol, chloroplast stroma and thylakoid lumen, and apoplast, it is transported throughout the cell and to the apoplast through specific transport across the chloroplast envelope and plasma membrane (Rautenkranz et al., 1994; Horemans et al., 1997). Following its use by ascorbate peroxidase in H2O2 detoxification, its participation in the Xanthophyll cycle, or its reduction of a-tocopherol radicals as part of non-photochemical quenching, ASC is oxidized to the monodehydroascorbate (MDHA) radical which disproportionates to ASC and dehydroascorbate (DHA). Although the rate of ASC synthesis is not fast, ASC is rapidly regenerated from DHA, a reaction catalyzed by DHAR and which uses glutathione as the reductant. Glutathione reductase uses NADPH produced principally from PSI to regenerate glutathione from oxidized glutathione. Consequently, the detoxification of AOS species by this ascorbate-glutathione pathway involves the transfer of electrons from PSI to NADPH to glutathione to ASC to H2O2 in a series of reactions in which there is no net loss of ASC or glutathione. Given its role in regenerating ASC, the principal function of DHAR activity would be expected to maintain the existing pool of ASC in a reduced state needed to meet the challenge imposed by those stresses that generate active oxygen species.
As an antioxidant, one of ASC's role in plants is to scavenge hydrogen peroxide. Hydrogen peroxide is involved in regulating the stomatal openings formed by the presence of pairs of guard cells in plants. Stomatal closure can be triggered by an increase in the intracellular concentration of hydrogen peroxide in guard cells. Stomatal openings can be triggered by a decrease in the intracellular concentration of hydrogen peroxide in guard cells. Plants control exposure to environmental conditions by tightly regulating stomatal aperture. Stomatal closures limits exposure to plants of certain toxins that may be circulating in the environment and protect the plant from drought conditions. Stomatal closures, however, also limit CO2 assimilation and increase the concentration of NADPH in plants as a consequence of reduction in Calvin cycle activity. Because of the importance of ASC levels in plants, there exists a need for closely regulating its production. This invention meets this and other needs.